Methods for lignocellulosic biomass pre-treatment: densification of biomass with alkaline or acidic chemical(s) thereof and for biotransformation thereof

ABSTRACT

A biomass pretreatment method is provided to improve the bio-digestibility of lignocellulosic biomass. This pretreatment method densifies biomass with alkaline or acidic chemical(s) to obtain densified biomass containing chemicals. Pretreatment effects are realized during densification. The chemicals in densified biomass further react with biomass during biomass storage and transportation to improve the pretreatment effects. Further treatment (e.g. stream) can also be applied to the densified biomass to achieve better effects. As chemicals are mixed well with biomass during densification and the biomass density is greatly increased, further treatment is of high efficiency and high solid loading. This pretreatment method is simple and the densified biomass with chemicals is resistant to microbial contamination/degradation and thus facilitates storage and transportation.

TECHNICAL FIELD

The invention belongs to the technical field of biorefining, which involves methods for pretreatment of lignocellulosic biomass by densification of biomass with alkaline or acidic reagents thereof and for biotransformation thereof.

BACKGROUND OF THE INVENTION

Lignocellulosic biomass is one of the most abundant renewable resources, which is widely distributed and easy to obtain. Biorefinery of lignocellulosic biomass for various bioproducts production can not only help improve energy security, boost rural economy, but also reduce greenhouse gas emission and promote sustainability.

Lignocellulosic biomass' adverse properties, such as low density and easy for mold to grow on, lead to high costs of transportation and storage, which to some extent impede the industrialization of lignocellulosic biomass refinery.

Pretreatment is an important step for conversion of lignocellulosic biomass to bioproducts, which disrupts plant cell wall structure and reduces the recalcitrance of lignocellulosic biomass for bioconversion. Traditional pretreatment methods have disadvantages such as severe reaction conditions, severe reactor corrosion, high energy consumption, etc (See Rezania S et al., Different pretreatment technologies of lignocellulosic biomass for bioethanol production: An overview, Energy. Sci., DOI: 10.1016/j.energy.2020.117457)(May 15, 2020). Furthermore, the low density of loose biomass limits biomass solid loading during pretreatment, leading to low productivity and high cost of pretreatment.

Densification of lignocellulosic biomass can increase biomass density to 5-15 folds and thus substantially reduces the costs of storage and transportation. Furthermore, the densified biomass owns the same shapes and sizes, which facilitates operations (See Zhang P F et al., Ultrasonic Vibration-Assisted Pelleting of Wheat Straw: A Designed Experimental Investigation on Pellet Quality and Sugar Yield, Asme International Manufacturing Science& Engineering Conference.Sci.,DOI: 10.1115/MSEC2010-34179 (January, 2012) and Theerarattananoon K et al., Effects of the pelleting conditions on chemical composition and sugar yield of corn stover, big bluestem, wheat straw, and sorghum stalk pellets, Bioprocess BiosystEng. Sci.,DOI: 10.1007/s00449-011-0642-8 (Oct. 11, 2012).

Densified biomass significantly increases the biomass loading in a reactor during pretreatment and thus increases pretreatment throughput (See the U.S. patent Ser. No. 10/730,958 B2, entitled, “Pretreatment of densified biomass using liquid ammonia and systems and products related thereto”). It also facilitates enzymatic hydrolysis, which follows pretreatment.

Although there are advantages of using densified biomass for storage, transportation and pretreatment, densified biomass is still easy to be contaminated by microbes and it requires severer conditions for pretreatment due to difficulties for mass and heat transfer. In particular, chemical reagents are difficult to penetrate into the densified biomass to achieve good pretreatment effect, which leads to extended pretreatment time or high pretreatment temperature.

DETAILED DESCRIPTION OF THE INVENTION

The present invention solves abovementioned issues related to transportation, storage and pretreatment of lignocellulosic biomass. This invention adds pretreatment chemical(s) (e.g. acid(s) and base(s)) into the biomass during densification. During the process, mechanical forces of densification and chemical reactions accelerated by the heat released during densification together treat the biomass. The chemicals in the densified biomass further react with the biomass components during transportation and storage to boost the pretreatment effects. This method fully utilizes the time of transportation and storage for pretreatment. Moreover, the densified biomass containing chemicals is resistible to microbial contamination, which together with the high density property facilitates biomass storage and transportation.

This invention avoids high energy consumption of pretreatment and high requirements for pretreatment reactors.

Further treatment (e.g. using stream) of the densified biomass containing chemical(s) can further enhance the pretreatment effects. As chemical(s) is mixed well with biomass during densification, mass transfer is not an issue during further treatment. Furthermore, adding water to the densified biomass containing base releases heat, which alleviates the heat transfer issue for pretreatment of densified biomass.

The invented pretreatment method is named as Densifying Lignocellulosic biomass with Chemical(s)(DLC). For DLC pretreatment, the chemicals can be added when biomass is being densified, or be added and mixed with biomass first and then densified with biomass, or be added, mixed with biomass and let it react with biomass for a certain time before densification.

The obtained DLC biomass can be used directly for bioconversion (e.g. can be used for enzymatic hydrolysis to degrade cellulose and hemicellulose in the biomass to glucose and xylose) or be further treated before bioconversion.

The lignocellulosic biomass can be used for DLC pretreatment includes agricultural residues, such as wheat straw, corn stover, rice straw, sorghum straw, soybean straw; woody biomass, such as wood pulp wastes, wood chips, softwood, hardwood; grasses, aquatic plants, algae and feces. The water/moisture content of the biomass can be used for DLC pretreatment is in the range of 0%-90%.

For DLC pretreatment, alkaline chemicals/reagents such as sodium hydroxide, calcium hydroxide, potassium hydroxide, sodium sulphite, sodium bisulfate, sodium acetate, sodium carbonate, sodium bicarbonate, sodium sulfate, ethylenediamine, triethylamine, ammonia, calcium oxide, sodium oxide, potassium oxide, etc. can be used.

For DLC pretreatment, acidic chemicals/reagents such as sulfuric acid, nitric acid, nitrous acid, hydrochloric acid, phosphoric acid, sulphurous acid, perchloric acid, chloric acid, chlorous acid, hypochlorous acid, oxalic acid, formic acid, acetic acid, propionic acid, benzoic acid, phenylacetic acid, benzenesulfonic acid, succinic acid, carbonic acid, carbon dioxide, sulfur dioxide, sodium bisulfate, sodium bisulfite, etc. can be used.

The dosage of alkaline or acidic chemicals/reagents for DLC pretreatment is 0.5° 4-30.0% of the biomass dry weight.

The abovementioned chemicals or their solutions can be used for DLC pretreatment. Chemicals or their solutions can be added, poured, sprayed, injected with steam or injected as gaseous form to the biomass before densification. Chemicals or their solutions can also be added simultaneously with biomass to the densification machine.

DLC pretreated biomass can be directly used for bioconversion, or be stored before bioconversion. DLC pretreated biomass can also be further treated before bioconversion. The further treatment can be spraying water on the biomass, steaming, soaking in water, sun exposure, freezing, high temperature treatment, cooking in water, high temperature sterilization, autoclave, microwave or ultrasonic treatment. Other lignocellulosic biomass pretreatment methods can also be used for further treatment, such as steam explosion, hot water pretreatment, steam pretreatment, dilute acid pretreatment, weak acid pretreatment, oxidative acid pretreatment, mixed acid pretreatment, dilute alkaline pretreatment, weak alkaline pretreatment, ammonia pretreatment, oxidative alkaline pretreatment, mixed alkaline pretreatment, or organic solvent pretreatment, etc.

The density of DLC pretreated biomass is in the range of 200-1500 kg/m³. The DLC pretreated biomass can be in shapes like pellets, briquettes, granular, cubes, etc.

Enzymes and microbes can be used for bioconversion of DLC pretreated biomass. Enzymes that can be used include cellulases, hemicellulases, pectinase, xylanase, alpha-amylase, glucoamylase. Microbes can be used to convert DLC pretreated biomass to bioporducts or be used with enzymes or be used in enzymatic hydrolysate of DLC pretreated biomass for bioproducts production. The microbes can be yeasts, bacteria, molds, etc.

Bioconversion of DLC pretreated biomass can produce fuels, bulk chemicals, fine chemicals, animal feed, food additives, and pharmaceuticals, such as ethanol, butanol, acetone, acetic acid, lactic acid, alkane, lipids, proteins, amino acids, enzymes, antibiotics, vitamins, antibodies, and methane.

The advantages of DLC pretreatment over other pretreatments include: (1) DLC pretreated biomass is much more suitable for storage and transportation; (2) DLC pretreatment integrates pretreatment with densification, reduces pretreatment cost; (3) DLC pretreatment conditions are mild and fully utilizes the time for biomass transportation and storage. (4) Further treatment, if needed, requires lower severity. (5) Due to the mild conditions used for DLC pretreatment, degradation products generated by DLC pretreatment might be fewer and less toxic, which facilitate bioconversion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a is the densified corn stover (without any chemical added during densification) at day 1.

FIG. 1b is the densified corn stover (without any chemical added during densification) stored at room temperature for 6 days.

FIG. 1c is the DLC pretreated corn stover with sodium hydroxide added during densification (NaOH dosage: 0.3 g/g dry biomass) at day 1.

FIG. 1d is the DLC pretreated corn stover with sodium hydroxide added during densification (NaOH dosage: 0.3 g/g dry biomass) stored at room temperature for 6 days.

FIG. 2a is showing microbes from a sample of loose corn stover are growing on a plate.

FIG. 2b is showing microbes from a sample of densified corn stover (without chemicals added during densification) are growing on a plate.

FIG. 2c is showing there is no microbe grew out from a sample of DLC pretreated corn stover (with sodium hydroxide added during densification).

FIG. 2d is showing there is no microbe grew out from a sample of DLC pretreated corn stover (with sulfuric acid added during densification).

FIGS. 3A&B are showing the sizes (diameter and length) of DLC pretreated corn stover used in example 2.

FIG. 4A is the loose corn stover in a 100 ml plastic cup.

FIG. 4B is the DLC pretreated corn stover (with sulfuric acid added during densification) in a 100 ml plastic cup.

FIG. 5 is showing the effect of storage time for DLC pretreated corn stover (with calcium hydroxide added during densification) on sugar release during enzymatic hydrolysis of DLC pretreated corn stover at 3% (w/w) solid loading.

FIG. 6 is showing the effect of storage time for DLC pretreated corn stover (with sodium hydroxide added during densification) on sugar release during enzymatic hydrolysis of DLC pretreated corn stover at 3% (w/w) solid loading.

FIG. 7 is showing sugar concentrations of hydrolysates derived from enzymatic hydrolysis of dilute acid (DA) pretreated corn stover and DLC followed by liquid hot water (LHW) treated corn stover at 10% (w/w) solid loading. Sulfuric acid (sa) was used for this DLC pretreatment. The pretreatment severity for DA and DLC(sa)+LHW is similar. Enzymatic hydrolysis was carried out for 72 h.

FIG. 8 is showing the sugar concentrations of hydrolysates derived from enzymatic hydrolysis of alkali (AL) pretreated corn stover (alkali used was sodium hydroxide(sh)), densified and then AL pretreated corn stover, DLC(sh) pretreated and then autoclaved corn stover. Enzymatic hydrolysis was performed at 6% (w/w) glucan loading for 24 h.

FIG. 9 is showing the sugar release with time during enzymatic hydrolysis of DLC followed by liquid hot water (LHW) treated corn stover, densified (pelletized) and then dilute acid pretreated corn stover, and dilute acid pretreated corn stover. Enzymatic hydrolysis was performed at 20% (w/w) solid loading. Sulfuric acid was used for this DLC pretreatment.

FIG. 10 is showing the fermentation performance of hydrolysate derived from enzymatic hydrolysis of DLC pretreated and then liquid hot water treated corn stover. Calcium hydroxide was used for DLC pretreatment. Enzymatic hydrolysis was conducted at 20% solid loading. No detoxification was applied.

FIG. 11 is showing the sugar concentrations of hydrolysates derived from enzymatic hydrolysis of DLC(sh) pretreated and then autoclaved corn stover at 3%, 6% and 9% glucan loading for 72 h.

DETAILED DESCRIPTION

The terms used here are the ones commonly used by people in this area and have the same meanings, unless otherwise mentioned.

The meanings of the abbreviations are listed as follows:

-   CS: corn stover; PCS: Pelletized Corn stover; DA: Dilute sulfuric     Acid pretreatment; AL: Alkaline pretreatment; LHW: Liquid Hot Water     treatment; DLC: Densifying Lignocellulosic biomass with Chemical(s)     pretreatment; DLC(ch): DLC pretreatment using calcium hydroxide;     DLC(sh): DLC pretreatment using sodium hydroxide; DLC(sa): DLC     pretreatment using sulfuric acid; PCS+AL: corn stover was pelletized     and then pretreated by AL; DLC(sa)+LHW: DLC(sa) pretreatment     followed by LHW treatment; DLC(sh)+Autoclave: DLC(sh) pretreatment     followed by autoclave; DLC(sh)+LHW: DLC(sh) pretreatment followed by     LHW treatment.

In the following examples, pelletization was used for densification.

Example 1

Example 1 is intended to show that DLC pretreated biomass avoids microbial contamination and thus is good for transportation and storage.

The corn stover was harvested, dried in the field, and then milled to 1-4 mm particles.

The milled corn stover was sprayed with sodium hydroxide solution or sulfuric acid. The dosage for sodium hydroxide was 0.3 g/g dry biomass. The dosage for sulfuric acid was 0.1 g/g dry biomass. Final water content was 0.5 g/g dry biomass. The corn stover was then pelletized to obtain DLC(sh) corn stover and DLC(sa) corn stover and then stored at room temperature for 6 days.

The milled corn stover was also pelletized without addition of any chemical to obtain PCS for control.

The PCS at day 1 is shown in FIG. 1a with no microbial contamination was observed. After 6 days storage at room temperature, microbial contamination was seen on the surface of the PCS (FIG. 1b ). On the contrast, microbial contamination on DLC(sh)-CS was not observed after 6 days storage (FIG. 1d ), which looked the same as day 1 (FIG. 1c ).

Loose CS, DLC(sh)-CS, DLC(sa)-CS and PCS were mixed with sterile water separately at a biomass to water ratio of 1:20. A liquid sample was taken from each mixture and plated on Luria-Bertani (LB) agar medium, which was then cultured at 30° C. for 3 days.

As seen in FIG. 2, the plate for loose CS grew out many microbial colonies (FIG. 2a ); there were also some microbial colonies grew out on the plate for PCS (FIG. 2b ), but there was no microbial colonies grew out on plates for DLC(sh)-CS (FIG. 2c ) and for DLC(sa)-CS (FIG. 2d ).

This experiment indicated that DLC pretreatment can not only kill microbes in the biomass but also help protect biomass from microbial contamination, which is important for guaranteeing a consistent and good quality of biomass and thus is also important for biomass storage and transportation.

Example 2

The corn stover was harvested, dried in the field, and then milled to 1-4 mm particles. The milled corn stover was sprayed with sodium hydroxide solution or sulfuric acid. The dosage for sodium hydroxide was 0.3 g/g dry biomass. The dosage for sulfuric acid was 0.1 g/g dry biomass. Final water content was 0.5 g/g dry biomass. The corn stover was then pelletized to obtain DLC(sh) corn stover and DLC(sa) corn stover.

The sizes of obtained DLC(sa) corn stover are shown in FIG. 3, with a diameter of around 0.8 cm, a length of about 2.5 cm and a mass of 1.85 g. The volume of this DLC(sa)-CS is

${{\pi*2.5*\left( \frac{0.8}{2} \right)^{2}} = {1.256\mspace{14mu}{mL}}},$

and density is

$\frac{1.85*1000}{1.256} = {1473\mspace{14mu}{kg}\text{/}{m^{3}.}}$

DLC(sh)-CS has similar sizes and density.

FIG. 4A shows a 100 ml plastic cup fully filled with 12.54 g loose corn stover. FIG. 4B shows the same cup fully filled with 81.81 g DLC(sa)-CS. Therefore, DLC(sa)-CS's bulk density is around 6.5 times of loose corn stover's, which indicates the advantage of DLC pretreated biomass in transportation and storage.

Example 3

This example shows the effect of storage time of DLC(ch) pretreated biomass on its enzymatic digestibility.

The corn stover was harvested, dried in the field, and then milled to 1-4 mm particles. The milled corn stover was sprayed with calcium hydroxide solution. The dosage of calcium hydroxide was 0.15 g/g dry biomass. The final water content was 0.5 g/g dry biomass. The corn stover was then pelletized and DLC(ch)-CS was obtained. The DLC(ch)-CS was stored at room temperature.

The DLC(ch)-CS stored for different days was enzymatically hydrolyzed at 3% (w/w) solid loading using cellulases and hemicellulases. Enzymatic hydrolysis was performed at 50° C. and 250 rpm in a shake incubator for 24 h using citrate buffer to control pH at 4.8.

As shown in FIG. 5, with the increase of storage time the sugar (glucose and xylose) released during enzymatic hydrolysis increased. The enzymatic hydrolysis of DLC(ch)-CS stored for 12 days released 6.9 g/L glucose and 2.8 g/L xylose.

This experiment indicated that pretreatment reactions were still occurring during storage of DLC-CS and storage and transportation time can be used to achieve a better pretreatment effect.

Example 4

This example shows the effect of storage time of DLC(sh) pretreated biomass on its enzymatic digestibility.

The corn stover was harvested, dried in the field, and then milled to 1-4 mm particles. The milled corn stover was sprayed with sodium hydroxide solution. The dosage of sodium hydroxide was 0.3 g/g dry biomass. The final water content was 0.5 g/g dry biomass. The corn stover was then pelletized and DLC(sh)-CS was obtained. The DLC(sh)-CS was stored for 2, 6.12 days at room temperature.

The DLC(sh)-CS stored for different days was enzymatically hydrolyzed at 3% (w/w) solid loading using cellulases and hemicellulases. Enzymatic hydrolysis was performed at 50° C. and 250 rpm in a shake incubator for 24 h using citrate buffer to control pH at 4.8.

As shown in FIG. 6, with the increase of storage time the sugar (glucose and xylose) released during enzymatic hydrolysis increased. The enzymatic hydrolysis of DLC(sh)-CS stored for 12 days released 8.0 g/L glucose and 3.0 g/L xylose.

This experiment also indicated that pretreatment reactions were still occurring during storage of DLC-CS and storage and transportation time can be used to achieve a better pretreatment effect.

Example 5

This example is to compare the enzymatic digestibility of DLC pretreated followed by liquid hot water treated corn stover (DLC(sa)+LHW-CS) and traditional dilute acid pretreated corn stover (DA-CS).

The corn stover was harvested, dried in the field, and then milled to 1-4 mm particles.

DLC(sa) pretreatment: The milled corn stover was sprayed with sulfuric acid. The dosage for sulfuric acid was 0.1 g/g dry biomass. The final water content was 0.5 g/g dry biomass. The corn stover was then pelletized to obtain DLC(sa) corn stover. The DLC(sa)-CS was stored in a plastic bag at room temperature for at least a day.

LHW treatment on DLC(sa)-CS: DLC(sa)-CS and water were added into a reactor, heated to 160° C. and maintained at 160° C. for 10 min before cooling down. The total water in the reaction system was 9 times of the dry weight of DCL(sa)-CS. As DLC(sa)-CS contained sulfuric acid, this treatment conditions were similar to traditional dilute acid pretreatment.

Traditional dilute acid pretreatment: loose corn stover, sulfuric acid and water were added into the reactor, heated to 160° C. and maintained at 160° C. for 10 min before cooling down. The sulfuric acid dosage was 0.1 g/g dry corn stover. The total water in the reaction system was 9 times of the dry weight of corn stover.

The pretreated corn stover was enzymatically hydrolyzed at 10% solid loading using cellulases and hemicellulases at 50° C. and 250 rpm in a shake incubator for 72 h. Citrate buffer was used to control pH at 4.8.

As shown in FIG. 7, enzymatic hydrolysis of DLC(sa)+LHW-CS released 40 g/L glucose, which was substantially higher than that obtained by enzymatic hydrolysis of DA-CS (28 g/L). Xylose released by enzymatic hydrolysis of DLC(sa)+LHW-CS reached 12 g/L, which was similar compared to DA-CS.

Comparing to DA pretreatment, DLC(sa)+LHW pretreatment used the same amount of sulfuric acid and the same reaction conditions during LHW treatment. The only additional operation is the biomass densification. Biomass densification saves a lot cost of biomass transportation and storage. Therefore, densification actually saves cost rather than adds cost.

As DLC(sa)+LHW pretreatment released much more sugar during enzymatic hydrolysis compared to DA pretreatment and does not add cost, DLC(sa)+LHW pretreatment seems to have more potential for industrial application.

Example 6

This example compares the enzymatic digestibility of alkaline pretreated corn stover (AL-CS), pelletized and then alkaline pretreated corn stover (PCS+AL-CS), and DLC(sh) pretreated followed by autoclaved corn stover (DLC(sh)+Autoclave-CS).

The corn stover was harvested, dried in the field, and then milled to 1-4 mm particles.

DLC(sh)+Autoclave treatment: The milled corn stover was sprayed with sodium hydroxide solution. The dosage of sodium hydroxide was 0.1 g/g dry biomass. The final water content was 0.5 g/g dry biomass. The corn stover was then pelletized and DLC(sh)-CS was obtained. The DLC(sh)-CS was stored in a sealed plastic bag at room temperature on a lab bench for at least a day. The DLC(sh)-CS was then autoclaved at 120° C. for 60 min in a shake flask with water addition to reach the DLC(sh)-CS solid loading of 30% (w/w).

PCS+AL pretreatment: the loose corn stover was pelletized and the pelletized corn stover was mixed with a sodium hydroxide solution to reach a solid loading of 20% (w/w). The mixture was then autoclaved at 120° C. for 120 min. The sodium hydroxide dosage was 0.1 g/g dry biomass.

AL pretreatment: the loose corn stover was mixed with a sodium hydroxide solution to reach a solid loading of 10% (w/w). The mixture was then autoclaved at 120° C. for 20 min. The sodium hydroxide dosage was 0.2 g/g dry biomass.

Enzymatic hydrolysis of pretreated corn stover was performed at 6% glucan loading using cellulases and hemicellulases at 50° C. and 250 rpm in a shake incubator for 24 h. 6% glucan loading refers to there is 6 g glucan in a total mixture of 100 g. Corn stover (un-pretreated) contains around 33% glucan.

The sugar concentrations obtained from enzymatic hydrolysis are shown in FIG. 8. DLC(sh)+Autoclave, PCS+AL and AL pretreatments resulted in similarly high sugar concentrations. However, the sodium hydroxide dosage for DLC(sh)+Autoclave and PCS+AL was 50% of that used for AL, and DLC(sh)+Autoclave had the highest solid loading for pretreatment. Furthermore, DLC(sh)+Autoclave used half of the autoclave time compared to PCS+AL. Although DLC(sh)+Autoclave treatment used 3 times more time compared to AL pretreatment, DLC(sh)+Autoclave treatment used 3 times higher solid loading. Moreover, DLC(sh) has the advantage of avoiding microbial contamination.

Example 7

This example compares the enzymatic digestibility of dilute acid pretreated corn stover (DA-CS), pelletized and then dilute acid pretreated corn stover (PCS+DA-CS), and DLC(sa) pretreated followed by liquid hot water treated corn stover (DLC(sa)+LHW-CS).

The corn stover was harvested, dried in the field, and then milled to 1-4 mm particles.

DLC(sa) pretreatment: The milled corn stover was sprayed with sulfuric acid. The dosage for sulfuric acid was 0.1 g/g dry biomass. The final water content was 0.5 g/g dry biomass. The corn stover was then pelletized to obtain DLC(sa) corn stover. The DLC(sa)-CS was stored in a sealed plastic bag at room temperature for at least a day before use.

LHW treatment on DLC(sa)-CS: the DLC(sa)-CS and water were added into a reactor, heated to 160° C. and maintained at 160° C. for 10 min. The final water in the reaction system was 5 times of the dry weight of DLC(sa)-CS.

PCS+DA pretreatment: the loose corn stover was pelletized and the pelletized corn stover was mixed with a sulfuric acid solution. The mixture was then heated to 160° C. and maintained at 160° C. for 10 min. The sulfuric acid dosage was 0.1 g/g dry corn stover. The final water in the reaction system was 5 times of the dry weight of PCS.

DA pretreatment: the loose corn stover was mixed with a sulfuric acid solution. The mixture was then heated to 160° C. and maintained at 160° C. for 10 min. The sulfuric acid dosage was 0.1 g/g dry corn stover. The final water in the reaction system was 9 times of the dry weight of CS.

Enzymatic hydrolysis of pretreated corn stover was performed at 20% solid loading using cellulases and hemicellulases at 50° C. and 250 rpm in a shake incubator for 72 h.

Sugar concentrations of enzymatic hydrolysis as a function of time are shown in FIG. 9. Glucose concentration for DLC(sa)+LHW-CS reached 73 g/L after 72 h enzymatic hydrolysis, which was higher than that for PCS+DA-CS (70 g/L) and for DA-CS (68 g/L). The xylose concentration for DLC(sa)+LHW-CS was also higher than the other two pretreated corn stover. Furthermore, the enzymatic hydrolysis rate of DLC(sa)+LHW-CS was also the highest among the tested three.

Example 8

This example is to test the fermentability of DLC(ch)+LHW-CS.

The corn stover was harvested, dried in the field, and then milled to 1-4 mm particles. The milled corn stover was sprayed with calcium hydroxide solution. The dosage of calcium hydroxide was 0.15 g/g dry biomass. The final water content was 0.5 g/g dry biomass. The corn stover was then pelletized and DLC(ch)-CS was obtained. The DLC(ch)-CS was stored in a sealed plastic bag at room temperature for at least a day before use.

LHW treatment on DLC(ch)-CS: The DLC(ch)-CS and water was added into a reactor, heated to 140° C. and maintained at 140° C. for 60 min. The water addition was 5 times of dry weight of DLC(ch)-CS.

The DLC(ch)+LHW-CS was enzymatically hydrolyzed at 20% (w/w) solid loading using cellulases and hemicellulases. Enzymatic hydrolysis was performed at 50° C. and 250 rpm in a shake incubator for 72 h. DLC(ch)+LHW-CS was all added at the beginning of the enzymatic hydrolysis (no fed-batch was applied).

The obtained enzymatic hydrolysate (containing 91.1 g/L glucose and 24.4 g/L xylose) was fermented using a bacterial strain, which can ferment both glucose and xylose to ethanol. The initial optical density at 600 nm (OD₆₀₀) for fermentation was 2.0. Fermentation was carried out in shake flasks at 30° C. and 150 rpm for 72 h.

As shown in FIG. 10, both glucose and xylose were fermented quickly with 51.5 g/L ethanol generated after 36 h. The total sugar conversion reached 97%. There was no detoxification during the whole process, which indicated high fermentability of DLC(ch)+LHW treated corn stover.

Example 9

This example is to show the effect of solid loading on enzymatic hydrolysis of DLC(sh)+Autoclave-CS.

The corn stover was harvested, dried in the field, and then milled to 1-4 mm particles.

DLC(sh)+Autoclave treatment: The milled corn stover was sprayed with sodium hydroxide solution. The dosage of sodium hydroxide was 0.1 g/g dry biomass. The final water content was 0.5 g/g dry biomass. The corn stover was then pelletized and DLC(sh)-CS was obtained. The DLC(sh)-CS was stored in a sealed plastic bag at room temperature for at least a day. The DLC(sh)-CS was then autoclaved at 120° C. for 60 min in a shake flask with water addition to reach the DLC(sh)-CS solid loading of 30% (w/w).

DLC(sh)+Autoclave-CS was enzymatically hydrolyzed at 3%, 6%, and 9% (w/w) glucan loading using cellulases and hemicellulases. Enzymatic hydrolysis was performed at 50° C. and 250 rpm in a shake incubator for 72 h.

As shown in FIG. 11, sugar concentrations increased with the increase of glucan loading. Glucose, xylose and total sugar reached as high as 105.1 g/L, 40.1 g/L and 145.2 g/L at 9% glucan loading. 

1. A method comprising: densifying lignocellulosic biomass with alkaline or acidic chemical(s)/reagent(s) to obtain densified lignocellulosic biomass containing chemical(s)/reagent(s).
 2. The method of claim 1 wherein the chemical(s)/reagent(s) are added when biomass is being densified, or are added and mixed with biomass first and then densified with biomass, or are added, mixed with biomass and reacted with biomass for a certain time before densification.
 3. A method comprising: using enzymes or/and microbe(s) to convert the densified lignocellulosic biomass prepared as in claim 1 or the densified lignocellulosic biomass prepared as in claim 1 and further treated to product(s).
 4. The method of claim 1 wherein the lignocellulosic biomass is selected from agricultural residues (e.g. wheat straw, corn stover, rice straw, sorghum straw, soybean straw), woody biomass (e.g. wood pulp wastes, wood chips, softwood, hardwood), grasses (e.g. switchgrass), aquatic plants, algae, feces and combinations thereof.
 5. The method of claim 1 wherein the water/moisture content of the biomass is in the range of 0%-90%.
 6. The method of claim 1 wherein the alkaline chemicals/reagents is selected from sodium hydroxide, calcium hydroxide, potassium hydroxide, sodium sulphite, sodium bisulfite, sodium acetate, sodium carbonate, sodium bicarbonate, sodium sulfate, ethylenediamine, triethylamine, ammonia, calcium oxide, sodium oxide, potassium oxide and combinations thereof.
 7. The method of claim 1 wherein the acidic chemicals/reagents is selected from sulfuric acid, nitric acid, nitrous acid, hydrochloric acid, phosphoric acid, sulphurous acid, perchloric acid, chloric acid, chlorous acid, hypochlorous acid, oxalic acid, formic acid, acetic acid, propionic acid, benzoic acid, phenylacetic acid, benzenesulfonic acid, succinic acid, carbonic acid, carbon dioxide, sulfur dioxide, sodium bisulfate, sodium bisulfite, and combinations thereof.
 8. The method of claim 1 wherein the dosage of alkaline or acidic chemical(s)/reagent(s) is in the range of 0.5%-30.0% of the biomass dry weight.
 9. The method of claim 1 wherein the chemical(s)/reagent(s) are used directly or are used to prepare their/its solution(s) before use.
 10. The method of claim 2 wherein the chemical(s)/reagent(s) or their solutions are added, poured, sprayed, injected with steam or injected as gaseous form to the biomass.
 11. The method of claim 3 wherein the further treatment of densified lignocellulosic biomass is selected from storage, transportation, spraying water on the biomass, steaming, soaking in water, sun exposure, freezing, high temperature treatment, cooking in water, high temperature sterilization, autoclave, microwave, ultrasonic treatment, other lignocellulosic biomass pretreatment methods, such as steam explosion, hot water pretreatment, steam pretreatment, dilute acid pretreatment, weak acid pretreatment, oxidative acid pretreatment, mixed acid pretreatment, dilute alkaline pretreatment, weak alkaline pretreatment, ammonia pretreatment, oxidative alkaline pretreatment, mixed alkaline pretreatment, organic solvent pretreatment, and combinations thereof.
 12. The method of claim 1 wherein the density of the densified lignocellulosic biomass is in the range of 200-1500 kg/m³.
 13. The method of claim 1 wherein the densified lignocellulosic biomass has a substantially cylindrical shape or a substantially rectangular shape.
 14. The method of claim 3 wherein the enzymes are selected from cellulases, hemicellulases, pectinase, xylanase, alpha-amylase, glucoamylase, and combinations thereof; the microbe(s) is selected from yeasts, bacteria, molds, and combinations thereof.
 15. The method of claim 3 wherein the product(s) is selected from fuels, bulk chemicals, fine chemicals, animal feed, food additives, pharmaceuticals, and combinations thereof, such as sugars, ethanol, butanol, acetone, acetic acid, lactic acid, alkane, lipids, proteins, amino acids, enzymes, antibiotics, vitamins, antibodies, and methane.
 16. The method of claim 3 wherein the lignocellulosic biomass is selected from agricultural residues (e.g. wheat straw, corn stover, rice straw, sorghum straw, soybean straw), woody biomass (e.g. wood pulp wastes, wood chips, softwood, hardwood), grasses (e.g. switchgrass), aquatic plants, algae, feces and combinations thereof. 